No abstract

The study of nuclear mechanical properties can provide insights into nuclear dynamics and its role in cellular mechanotransduction. While several methods have been developed to characterize nuclear mechanical properties, direct intracellular probing of the nucleus in situ is challenging. Here, a modified AFM (atomic force microscopy) needle penetration technique is demonstrated to mechanically characterize cell nuclei in situ. Cytoplasmic and nuclear stiffness were determined based on two different segments on the AFM indentation curves and were correlated with simultaneous confocal Z-stack microscopy reconstructions. On the basis of direct intracellular measurement, we show that the isolated nuclei from fibroblast-like cells exhibited significantly lower Young's moduli than intact nuclei in situ. We also show that there is in situ nucleus softening in the highly metastatic bladder cancer cell line T24 when compared to its less metastatic counterpart RT4. This technique has potential to become a reliable quantitative measurement tool for intracellular mechanics studies.

The physical forces that drive morphogenesis are not well characterized in vivo, especially among vertebrates. In the early limb bud, dorsal and ventral ectoderm converge to form the apical ectodermal ridge (AER), although the underlying mechanisms are unclear. By live imaging mouse embryos, we show that prospective AER progenitors intercalate at the dorsoventral boundary and that ectoderm remodels by concomitant cell division and neighbour exchange. Mesodermal expansion and ectodermal tension together generate a dorsoventrally biased stress pattern that orients ectodermal remodelling. Polarized distribution of cortical actin reflects this stress pattern in a β-catenin- and Fgfr2-dependent manner. Intercalation of AER progenitors generates a tensile gradient that reorients resolution of multicellular rosettes on adjacent surfaces, a process facilitated by β-catenin-dependent attachment of cortex to membrane. Therefore, feedback between tissue stress pattern and cell intercalations remodels mammalian ectoderm.

Topographic nanomanufacturing with a depth precision down to atomic dimension is of importance for advancement of nanoelectronics with new functionalities. Here we demonstrate a mask-less and chemical-free nanolithography process for regio-specific removal of atomic layers on a single crystalline silicon surface via shear-induced mechanochemical reactions. Since chemical reactions involve only the topmost atomic layer exposed at the interface, the removal of a single atomic layer is possible and the crystalline lattice beneath the processed area remains intact without subsurface structural damages. Molecular dynamics simulations depict the atom-by-atom removal process, where the first atomic layer is removed preferentially through the formation and dissociation of interfacial bridge bonds. Based on the parametric thresholds needed for single atomic layer removal, the critical energy barrier for water-assisted mechanochemical dissociation of Si–Si bonds was determined. The mechanochemical nanolithography method demonstrated here could be extended to nanofabrication of other crystalline materials.

Wnt signaling plays a key role in embryogenesis and cancer development. Dvl (Dishevelled) is a central mediator for both the canonical and noncanonical Wnt pathways. Dact1 (Dapper1, Dpr1), a Dvl interactor, has been shown to negatively modulate Wnt signaling by promoting lysosomal degradation of Dvl. Here we report that Dact1-deficient mice have multiple physiological defects that resemble the human neonate disease congenital caudal regression syndrome, including caudal vertebrae agenesis, anorectal malformation, renal agenesis/dysplasia, fused kidneys, and loss of bladder. These urogenital defects can be traced to impaired hindgut formation starting at embryonic day 8.25. Examination of morphological changes and Wnt target gene expression revealed that the planar cell polarity (PCP) signaling is deregulated, whereas the canonical Wnt/␤-catenin pathway is largely unaffected in mutant embryos. Consistently, the activity of the PCP signal mediators Rho GTPase and c-Jun N-terminal kinase is altered in Dact1 ؊/؊ mouse embryonic fibroblasts. We further observed alterations in the protein level and the cellular distribution of Dvl in the primitive streak of mutant embryos. An increased amount of Dvl2 tends to be accumulated in the cortical regions of the cells, especially at the primitive streak ectoderm close to the posterior endoderm that lately forms the hindgut diverticulum. Together, these data suggest that Dact1 may regulate vertebrate PCP by controlling the level and the cellular localization of Dvl protein.